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WO2009114869A2 - Procédés de préparation et composition de constructions de peptide utiles pour le traitement de la polyarthrite rhumatoïde - Google Patents

Procédés de préparation et composition de constructions de peptide utiles pour le traitement de la polyarthrite rhumatoïde Download PDF

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Publication number
WO2009114869A2
WO2009114869A2 PCT/US2009/037312 US2009037312W WO2009114869A2 WO 2009114869 A2 WO2009114869 A2 WO 2009114869A2 US 2009037312 W US2009037312 W US 2009037312W WO 2009114869 A2 WO2009114869 A2 WO 2009114869A2
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seq
peptide
cells
group
subset
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PCT/US2009/037312
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WO2009114869A3 (fr
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Daniel H. Zimmerman
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Cel-Sci Corporation
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Priority to EP09718824.7A priority Critical patent/EP2254588B1/fr
Priority to US12/922,687 priority patent/US11041013B2/en
Publication of WO2009114869A2 publication Critical patent/WO2009114869A2/fr
Publication of WO2009114869A3 publication Critical patent/WO2009114869A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C07K14/7055—Integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/008—Leishmania antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention is related to peptide constructs, i.e., polypeptides obtained by linking together two or more peptides based on or derived from different molecules, which are useful in the treatment or prevention of autoimmune diseases, particularly rheumatoid arthritis, asthma, allergies, and host versus graft (or graft versus host) rejection, as well as to compositions containing same, methods for producing same and methods for using same.
  • peptide constructs i.e., polypeptides obtained by linking together two or more peptides based on or derived from different molecules, which are useful in the treatment or prevention of autoimmune diseases, particularly rheumatoid arthritis, asthma, allergies, and host versus graft (or graft versus host) rejection, as well as to compositions containing same, methods for producing same and methods for using same.
  • HLA Leukocyte Antigens genotypes, have been identified, including those associated with Insulin Dependent Diabetes Mellitis (IDDM), Rheumatoid Arthritis (RA) (e.g. collagen type II 390-402 IAGFKGEQGPKGE (SEQ ID NO.l), Systemic Lupus Erythematousis (SLE), Ankyosing Spondylitis (AS), Pemphius Vulgaris (PV) (epidermal cell adhesion molecule desmoglein 190-204), Multiple Sclerosis (MS), Myelinproteolipid (MPL) (peptide sequence KNIVTPRT (SEQ ID NO.2), certain types of psoriasis, and uveoretintis (Hammer et ah, HLA class I peptide binding specificity and autoimmunity.
  • IDDM Insulin Dependent Diabetes Mellitis
  • RA Rheumatoid Arthritis
  • SLE Systemic Lupus Erythematousis
  • peptides are known that induce in animals, a condition similar to ones found in humans, such as GDKVSFFCKNKEKKC (SEQ ID NO.3) for antiphospholipid antibodies associated with thrombosis (Gharavi et ah, GDKV-Induced Antiphospholipid Antibodies Enhance Thrombosis and Activate Endothelial Cells In Vivo and In Vitro 1999, J.Immunol., 163:2922) or myelin peptides for experimental autoimmune encephalitis (EAE)as a model for MS (Ruiz et ah, supra, Araga et ah, A Complementary Peptide Vaccine That Induces T Cell Anergy and Prevents Experimental Allergic Neuritis in Lewis Rats 1999, J.
  • GAD glutamic acid decarboxylase
  • specific peptides have been identified associated with IDDM (Tisch et al, supra; Yoon et al., supra). Many of these conditions are also characterized by elevated levels of one or more different cytokines and other effectors such as Tumor Necrosis Factor (TNF) (Kleinau et al., Importance of CD23 for Collagen-Induced Arthritis: Delayed Onset and Reduced Severity in CD23 -Deficient Mice 1999, J. Immunol.
  • TNF Tumor Necrosis Factor
  • CTLA-4 Cytotoxic T-lymphocyte-Associated protein 4
  • Efforts are underway to attack cells or cellular products of the immune system and thereby treat autoimmune conditions, allergies, asthma and transplantation rejection using as reagents presumptive antigenic peptides or proteins, peptides representing certain T cells, monoclonal antibodies (Mabs) or recombinant proteins binding various effector cells or molecules such as Tumor Necrosis Factor Alpha (TNF ⁇ ) and IgE.
  • TNF ⁇ Tumor Necrosis Factor Alpha
  • LFA-3TIP fusion protein LFA-3TIP
  • AmeviveTM from Biogen
  • LFA-3TIP is bi-functional (i.e., two identical LFA-3 regions and TIP) and therefore is a complex conjugate molecule.
  • LFA-3TIP is a recombinant fusion protein designed to modulate the immune response by blocking the cellular pathway that activates T cells.
  • the compound is acting on a subset of memory effector cells with a down modulation or re-direction of modulation activity.
  • antigen non-specific approaches also utilize monoclonal antibodies that act on activated T cells and down regulate them such as using anti-CD3 (Protein Design Laboratories) or blocking Antigen Presenting Cells (APC) and T cell interaction by anti- Intercellular Adhesion Molecule 3 ((ICAM-3) (ICOS)).
  • ICM-3 Intercellular Adhesion Molecule 3
  • MEDI- 507 Medimmune
  • MEDI- 507 Medimmune
  • Other diseases such as tissue transplantation rejection and allergies are also being tested by this approach.
  • E25 (Genentech) binds to circulating IgE with the goal of preventing activation of mast cells.
  • rhu-Mab-E25 is believed to be a humanized monoclonal antibody against IgE.
  • Other researchers are developing monoclonal antibodies to act directly on agents causing disease symptoms.
  • Remicade Infliximab (Centocor) is purported to be a monoclonal antibody to TNF ⁇ while anti CD40 ligand has been used for treatment in animal model of MS (Howard et al., supra).
  • a recombinant generated designed protein Enbrel (Immunex) is purported to comprise two molecules of r-DNA derived TNF ⁇ soluble receptor, and is intended to block TNF ⁇ 's action.
  • GALT Gut Associated Lymphoid Tissues
  • a related approach to treat autoimmune conditions is the use of an oral formulation of peptide(s) as immunogen given in large quantities.
  • the peptide represents a sequence that is thought to be the autoimmune epitope itself or a modified form which may have altered binding or improved stability properties.
  • APL Altered Peptide Ligand
  • TCR T cell receptor
  • APC antigen presenting cell
  • MHC MHC
  • T cell receptor (TCR) molecules have also been contemplated by the prior art (Clay et ah, Changes in the Fine Specificity of gplOO (209 - 2 i 7) -Reactive T Cells in Patients Following Vaccination with a Peptide Modified at an HLA- A2.1 Anchor Residue 1999, J. Immunol., 162:1749).
  • a one amino acid change on the ⁇ -chain can abolish its oral immune tolerance activity in two (2) mechanistically different IDDM murine models (Homann et ah, Insulin in Oral Immune "Tolerance”: A One- Amino Acid Change in the B Chain Makes the Difference 1999, J.
  • truncated peptides of autoimmune inducing epitope are used as an antagonist in an animal model to treat a particular condition (Karin et al. supra).
  • Synthetic amino acid polymers that are thought to represent epitopes which contain Tyrosine (Y), Glutamic acid (E), alanine (A) and lysine (K) to target T cells such as Copolymer 1 have been contemplated.
  • Copaxone has been used as an oral tolerance delivery approach to treat MS patients where Copaxone is believed to be a synthetic copolymer of four amino acids (Hafler et al., supra 1988, J. Immunol., 141:131).
  • Modified peptides of peptide epitopes are also being studied for treatment of various autoimmune conditions including MS and PV (desmoglein-3) (Hammer, et al., supra 1997, Adv. Immunol., 66:67; Wucherpfennig et al., Structural Basis for Major Histocompatibility Complex (MHC)-Linked Susceptibility to Autoimmunity: Charged Residues of a Single MHC Binding Pocket Confer Selective Presentation of Self-Peptides in Pemphigus Vulgaris 1995, PNAS, 92:11935).
  • MS and PV desmoglein-3
  • MHC Major Histocompatibility Complex
  • myelin proteolipid associated peptide epitope a polymer or derivative of this epitope for MS has been further contemplated (Hammer et al., supra 1997, Adv. Immunol., 66:67).
  • peptides that are unique to the T cell antigen receptor molecule have been contemplated for a psoriasis vaccine such as IR 502 while others have been contemplated for Rheumatoid Arthritis.
  • T cell antigen Receptor TCRaVX
  • TCRaVX T cell antigen Receptor
  • Still another peptide approach uses complimentary peptide vaccine that induces T cell anergy and prevents EAE in rats by induction of anti-TCR antibodies (a Ia antiidiotype) and thereby elimination of these cells (Araga et al., supra 1999, J. Immunol., 163:476).
  • AD Alzheimer's Dementia
  • a ⁇ amyloid beta
  • passive agents as known in the art are composed of Mabs targeting cytokines, or cell surface marker, or soluble receptor with similar binding specificity.
  • cytokines or cell surface marker
  • soluble receptor with similar binding specificity.
  • a problem encountered with “passive agents” is that they must be frequently administered parenterally for the life of the patient.
  • the Mabs or soluble receptor must be “humanized” (huMab) to allow long term administration and to avoid potential long term adverse effects.
  • the production and sale of Mabs comprises one of the largest markets for treating chronic long-term conditions in the developed world.
  • agents for treatment ranges from insulin for type
  • a ⁇ Mabs are being investigated as potential products by a number of private corporations.
  • one known A ⁇ Mab investigated by Lilly is effective in decreasing A ⁇ burden in the cerebral cortex of "AD" mice.
  • This finding confirms previous reports of beneficial effects of plaque reduction in "AD" transgenic mice by other known Ab both Mab and Pab investigated by Elan (Bard et al., Epitope and isotype specificities of antibodies to beta-amyloid peptide for protection against Alzheimer's disease-like neuropathology, Proc Natl Acad Sci U.S.A. 2003 Feb 18;100(4):2023-8; DeMattos et al.
  • Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a mouse model of Alzheimer's disease, Proc Natl Acad Sci U.S.A. 2001 JuI 17;98(15):8850-5).
  • the necessity for inclusion of the Fc region was shown to be required in one compound entitled 3D3 which was found in the Central Nervous System (CNS) (Bard et al.).
  • the Mab 3D3 was mediated by binding to phagocytes. It was shown by the art that 3D3 (IgG2b) and 10D5 (IgGl) were effective in reducing A ⁇ plaques but not 16Cl 1 (IgGl) and 21F12 (IgG2a). Both IgG2a and IgG2b subclasses are considered as complement fixing antibodies that bind to Fc receptors. Furthermore, an association between A ⁇ plaque reduction and affinity of the Mabs was not validated.
  • Mab m266 is believed to act on CNS A ⁇ levels by serving as a sequestering agent acting from its plasma location given that it is not found in the CNS (DeMattos et al). It appears from the art that m266, 3D3 and 10D5 act by different mechanisms in plaque clearance wherein all three Mabs act as A ⁇ sinks in dialysis assays.
  • active agents in the treatment of A ⁇ , the objective is to induce the body to produce molecules to counteract or neutralize the agent.
  • active agents can be considered vaccines that induce a long lived adaptative immune response in patients through induction of antibodies and/or by Cytotoxic Lymphocytes (CTL) or other cellular mechanisms to clear or impede the deposition of A ⁇ .
  • CTL Cytotoxic Lymphocytes
  • virus like particles also suffer from the same disadvantages noted for viral vector vaccines whereby the particles incorporate antigenic epitope(s) of the plant, animal or bacterial virus (plasmid) where it is expressed in the VLP containing the foreign antigen of interest perhaps in a fusion protein along with the epitopes of the host virus or plasmid.
  • Another disadvantage in using a DNA-based vaccine including for autoimmune conditions is the possibility of the vaccine DNA being integrated into the host's genome.
  • One alternative is to conjugate a particular epitope to a carrier protein to avoid such incorporation into the genome.
  • KLH Keyhole Limpet Hemocyanin
  • BSA Bovine Serum Albumin
  • HSP Antigenics' heat shock proteins
  • peptide delivery of peptide epitopes include the use of synthetic biodegradable microparticles like Poly (lactide-co-glycolide) PLG with aggregated antigen. Still other delivery technologies for peptide antigens include Auto VacTM of Pharmexa. Other small molecule delivery technologies for peptides are Antigen Express's 'Ii-Key' delivery, phage display and Multiple Antigen Presentation (MAPS) technologies (Rosenthal 2005 Immune peptide enhancement of peptide based vaccines Frontiers in Bioscience 1:478-482).
  • MAMS Multiple Antigen Presentation
  • the therapeutic be a small peptide used as a potential antigen as in Epimmune's PadreTM or CEL-SCF s Ligand Epitope Antigen Presenting System (L.E.A.P.S.TM ) technology as described in U.S. 5,652,342, the contents of which are incorporated herein by reference.
  • L.E.A.P.S.TM Ligand Epitope Antigen Presenting System
  • L.E.A.P.S. uses small peptides referred to as T cell Binding Ligands (TCBL' s) as a peptide antigen delivery technology wherein the TCBL' s are peptide sequences derived from the human immune system molecules known or suspected to bind to human T cells.
  • TCBL' s T cell Binding Ligands
  • L.E.A.P.S. includes a first peptide which is an antigenic peptide associated with disease or the causative organism of disease covalently bonded to a second peptide which is a T cell binding ligand
  • the hetero-functional cellular immunological reagents taught by U.S. 5,652,342 are not antigen (disease) specific in certain cases. Instead, U.S.
  • T cell binding ligands including portions of MHC Classes I and II or accessory molecules such as ⁇ -2-microglobulin, portions of LFA-3, portions of the Fc region of the heavy chain of immunoglobulins, and Ia + molecules and generally teaches for the antigens associated with auto-immunity such as IDDM, RA and Thyroiditis, further the patent fails to provide more antigen (disease) specific treatment.
  • the therapeutics should also allow for the option of subcutaneous, oral intradermal, intranasal or transdermal administration wherein the active portion of the therapeutics should be comprised of substantially smaller molecules (3-5000Da) that are more likely to be able to cross the blood brain barrier.
  • the present invention provides peptide constructs useful for treatment of autoimmune disease, particularly for rheumatoid arthritis, asthma, allergy, and tissue transplantation rejection (including both host-versus-graft and graft-versus-host rejection), which differ from the above approaches used with antigenic peptide alone.
  • the novel constructs bind in an antigen specific manner and redirect the T cell in the direction of a non-deleterious autoimmune response, primarily from a ThI to a Th2 immune response, but where advantageous, primarily from a Th2 to a ThI immune response.
  • the novel constructs include one peptide component which will bind to T cells associated with autoimmune disease, asthma, allergies or host versus graft or graft versus host rejection while a second peptide component will bind to sites on the T cells which will preclude the normal sequence of events required for cell activation thereby initiating an abortative T cell modulation resulting in cell anergy and apoptosis.
  • novel peptides of this invention include peptide constructs of the following formula (I):
  • P 1 is a peptide associated with autoimmune disease, allergy or asthma, or tissue transplantation rejection and which will bind to an antigen receptor on a set or subset of T cells
  • P 2 is an immune response modifying peptide which will (i) cause a directed immune response by said set or subset of T cells to which the peptide P 1 is attached or (ii) bind to a T cell receptor which will cause said set or subset of T cells to which the peptide P 1 is attached to initiate, but not complete, an immune response causing said set or subset of T cells to undergo anergy and apoptosis
  • x is a direct bond or linker for covalently bonding P 1 and P 2 .
  • the present invention also provides a first method for treating or preventing inappropriate autoimmune response in individuals at risk for autoimmune disease, allergic reactions, asthma or host-graft or graft-host rejection, wherein a pharmacologically effective amount of a peptide construct of formula (I) is administered to the individual to effectively eliminate the set or subset of T cells involved in the autoimmune response.
  • the present invention also provides a second method for modulating an inappropriate autoimmune response in individuals at risk for autoimmune disease, allergic reactions, asthma or host-graft or graft-host rejection, wherein a pharmacologically effective amount of a peptide construct of formula (I) is administered to the individual to redirect the autoimmune response from a ThI to a Th2 immune response, or from a Th2 to a ThI immune response, whereby the inappropriate autoimmune response is modulated to decrease or eliminate the adverse effects associated with the inappropriate autoimmune response.
  • a pharmacologically effective amount of a peptide construct of formula (I) is administered to the individual to redirect the autoimmune response from a ThI to a Th2 immune response, or from a Th2 to a ThI immune response, whereby the inappropriate autoimmune response is modulated to decrease or eliminate the adverse effects associated with the inappropriate autoimmune response.
  • Fig. 1 represents data from mice received two treatments of 33nmoles (for CEL-
  • CEL-2000 2000 equivalent to 4.8mg/kG) of CEL-2000, (the same dose as used for the L.E.A.P.S. HSV vaccines), or other conjugates of the same collagen peptide used in CEL-2000 (SEQ ID NO. 852) using the same ICBLs as in the HSV or EAM systems for comparison using a GMP grade ICFA adjuvant.
  • Fig. 2 represent a study where CEL-2000 treatment with 2 doses of 33 or 100 nmol (the dose used in the EAM study) was given subcutaneously on days 0 and 7 as in the EAM study or 0 and 14 as for the HSV. Most regimes reduced the progression of arthritis disease to levels that were at least as good as those of mice treated with Enbrel (every other day for the 28 days of the study). Immunization of mice with the lOOnmol dose (3X treatment) on days 0 and 7 appeared to limit the progression of disease throughout the experimental period.
  • the CEL-2003 links the murine CII 254 - 273 sequence to the J ICBL.
  • Fig. 3 represents CIA Model analysis of foot pad thickness as evidence of CEL-
  • Fig. 4 represents a summary plot of the analysis performed cores of the 4 tissue areas, pannus membrane, inflammation, bone and cartilage from the following groups: Control no disease; CIA disease: CIA Disease Enbrel daily for 14 days continued for 2 more weeks before termination: CEL-2000 and CEL-2001 groups (vaccinated on day 0 and 7) from study 1 (Fig. 1) and from study 2 (Fig. 2) IX and 3X CEL-2000 vaccinated groups on day 0 and 7.
  • TCR ICAM- 1 also known as CD54
  • LFA-I also known as CDl la/CD 18
  • such rearrangement is prevented by the close association in a peptide construct using a TCBL from ICAM-I, LFA-3 (aa26- 42), VLWKKQKDKVAELENSE (SEQ ID NO.4) (Osborn et al, Amino acid residues required for binding of lymphocyte function- associated antigen 3 (CD58) to its counter- receptor CD2 1995, JEM, 181:429), by either the disparity in the temporal binding or higher strength of binding activity, thereby preventing the rearrangements and other more intimate interactions necessary for activation. Initially these sites are close together but normally rearrangements on the T cell surface occur during the activation process so by preventing this shift activation should not occur.
  • TCBL from CD4 that binds to the TCR and CD3 may be used as the TCBL in the peptide construct of this invention. Its binding to the T cell recognition site will inhibit subsequent events from occurring (MHC ⁇ with CD4 or ⁇ -2 with CD8).
  • Still another approach is a construct which redirects the immune response initiated by the natural autoimmune inducing event from a THl to a TH2 response ⁇ e.g., Lowrie et al., Therapy of tuberculosis in mice by DNA vaccination 1999, Nature, 400:269; Tisch et al., supra 1999, J. Immunol., 163:1178).
  • a TH2 directed response is one which directs the immune response toward the TH2 direction, thus favoring production of TH2 associated cytokines IL-5, IL-4, IL-IO, IL-13 TNF- ⁇ and antibody isotypes IgGl and IgG3 in mice (or comparables in man) as opposed to ThI, where the immune response favors production of cytokines IFN- ⁇ , IL-2, IL-6, IL- 12 cytokines and antibody isotypes IgG2a and IgG2b in mice and Cytotoxic T cell activity. It is understood, of course, that a "TH2 directed response" is not intended to imply an exclusively TH2 response, but rather a mixed immune response which is weighted to favor a TH2 profile.
  • a TCBL associated with TH2 responses e.g., peptide G from MHC class II (Zimmerman et ah, A new approach to T cell activation: natural and synthetic conjugates capable of activating T cells 1996, Vacc.
  • IL-4 or IL-5 or peptides known to stimulate IL-4 or IL-5 synthesis are used as the TCBL along with the autoimmune inducing peptide ⁇ e.g., Hammer et ah, supra, Krco et ah, supra, Araga et ah, supra, Ota et ah, supra, Ruiz et ah, supra, Yoon et ah, supra, Dittel et ah, Presentation of the Self Antigen Myelin Basic Protein by Dendritic Cells Leads to Experimental Autoimmune Encephalomyelitis 1999, J.
  • a TCBL such as peptide J, DLLKNGERIEKVE (SEQ ID NO. 51) (Zimmerman et ah, supra; Rosenthal et ah, supra) or ones known to stimulate IL-2 or IL- 12 synthesis would be used along with the autoimmune inducing peptide.
  • Yet another approach is to use the peptide construct to not activate the normal immune process but to activate the process leading to apoptosis of the T cell by using as the TCBL a ligand that binds to a site on the T cell whose normal binding and activation leads to apoptosis of the T cell; such as the TNF- receptor of the T cell, in which the TCBL would be the TNF- ⁇ ligand portion.
  • TNF peptides known to activate macrophages are amino acids 70-80 PSTHVLITHTI (SEQ ID NO.
  • H4-1-BB ligand is useful as a treatment for autoimmune disease similar to uses for flt3-L and CD40L; therefore, H4-1BB may also be used as TCBL for inclusion with autoimmune antigens to form the inventive peptide construct.
  • Other such TCBL examples are available from application with Fas and Fas-ligand including the noncleavable Fas-ligand (WO 99/36079A1).
  • Fas-Ligand or the sequence obtained by reverse engineering technique to determine amino acid (aa) sequence acting as receptor for DEVD-aldehyde or YVAD (SEQ ID NO. 22) chloromethylketone, may also be used as the TCBL.
  • IFN- ⁇ and the IFN- ⁇ ligand can also be used as TCBL' s in the invention peptide constructs.
  • the antigenic peptide and the peptide for T cell binding may be directly linked together in any order (i.e., N-terminal of one to C-terminal of other or vice versa) or the peptide may be covalently bonded by a spacer or linker molecule.
  • linkers between the two domains suitable examples include a thioether bond between an amino terminus bromoacetylated peptide and a carboxyl terminus cysteine, often preceded by a diglycine sequence (Zimmerman et ah, supra), carbodiimide linkages, a multiple glycine, e.g., from 3 to 6 glycines, such as triglycine, with or without one or two serines, separation between the two entities, e.g., GGGS (SEQ ID NO.7), GGGSS (SEQ ID NO. 8), GGGGS (SEQ ID NO.9), GGGGSS (SEQ ID NO. 10), GGGSGGGS (SEQ ID NO.11), etc., and other conventional linkages, such as, for example, the direct linkages such as, EDS, SPDP, and MBS, as disclosed in the aforementioned U.S. 5,652,342.
  • GGGS SEQ ID NO.7
  • GGGSS SEQ ID
  • P 1 is a peptide associated with autoimmune disease, allergy or asthma, or transplantation rejection and which will bind to an antigen receptor on a set or subset of T cells;
  • P 2 is an immune response modifying peptide which will bind to T cells to cause a directed immune response by said set or subset of T cells to which the peptide P 1 is attached or which will bind to a T cell receptor which will cause said set or subset of T cells to which the peptide P 1 is attached to initiate, but not complete, an immune response causing said set or subset of T cells to undergo anergy and apoptosis; and x is a direct bond or linker for covalently bonding P 1 and P 2 .
  • the TCBL portion of the immunomodulatory peptide construct of this invention i.e., P 2
  • an eight amino acid group LRGGGGSS (SEQ ID NO.12), of 11.2 Angstroms in length (Reineke et al., A synthetic mimic of a discontinuous binding site on interleukin-10 1999, Nature Biotechnology, 17:271) has been used to form a single peptide from two smaller discontinuous peptides of IL-IO, thereby forming a TCBL which could be used for redirection from a THl to a TH2, in combination with, for example, the IDDM, PV or MS inducing epitopes (Hammer et al., supra Tisch et al., supra).
  • Linkers (X) of varying lengths to form a single chain may be used, for example,
  • GGGS SEQ ID NO. 7
  • GGGGS SEQ ID NO. 9
  • Such linkers may result in a tertiary structure which might be of use to form a more avid TCBL (Shan et al., supra).
  • the peptide constructs of this invention may have as many as about 200 amino acids in its sequence, preferably up to about 150 amino acids, and especially, up to about 100 amino acids.
  • the minimum number of amino acids is also not strictly limited but usually each of the peptide components P 1 and P 2 will have at least about 4, preferably at least about 6, and more preferably at least about 8 or 9 amino acids in order to provide the appropriate epitope configuration for effectively binding to the appropriate site on the T cells of interest.
  • the peptide constructs of this invention will usually contain from about 20 to about 100 or more amino acids.
  • the peptide constructs may be prepared using conventional solid state peptide synthesis, provided however, that for constructs having more than about 40 amino acids, especially more than about 50 amino acids, it is usually convenient and preferred to prepare shorter segments and then link the shorter segments using well known techniques in solid phase peptide synthesis.
  • the peptide constructs may be prepared using conventional solution phase condensation chemistry of smaller peptides prepared by solid phase peptide synthesis, provided however, that for constructs having more than about 40 amino acids, especially more than about 50 amino acids, it is usually convenient and preferred to prepare shorter segments and then link the shorter segments using well known techniques in solid phase peptide synthesis.
  • the peptide constructs of this invention may be prepared using well known genetic engineering methods. Further details on methods for producing the instant peptide constructs can be found in the aforementioned U.S. 5,652,342.
  • Improved versions of the peptide constructs are comprised of variables X 1 to X 12 substitutions where each of X 1 to X 12 describe a group of particular types of amino acids based on their features.
  • amino acids can be nonpolar, hydrophobic while another group of amino acids are polar, uncharged amino acids that are hydrophilic.
  • amino acids can be grouped as those that are polar, charged, hydrophilic amino acids further subdivided between acidic and basic amino acids. Table 1 describes the chemical and structural features along with the corresponding amino acids.
  • Table 2 describes the amino acids for X 1 to X 10 wherein the amino acids for X 1 to X 1O have been selected on their common chemical and structural features as shown in Table 1 for inclusion in the improved variants of the invention.
  • Instances of X 2 X 3 or X 3 X 2 or X 2 X 3 or X 3 X 2 or X 3 X 3 or X 2 X 2 can be replaced by X 11 or by gamma amino butyric acid (GABA) wherein the common feature is that the amino function is on the ⁇ - carbon and not the ⁇ -carbon.
  • GABA gamma amino butyric acid
  • X 1 X 1 or X 1 X 3 or X 3 X 1 or X 1 X 2 or X 2 X 1 can be replaced by X 1S .
  • Modifications for increased stability at the amino terminus by use of an acetyl or propionyl group, D glycine or D alanine or use of cyclohexylalanine at the amino terminus to reduce proteolysis are contemplated by the substitution of X 12 .
  • X 12 can be present or not present on the sequence. It is noted that the sequences in computer readable form do not include X 12 . However, the presence of the modification is present for all sequences where disclosed in the instant specification.
  • X 13 modifications by use of 5-aminopentanoic acid for replacement of lengths of 3 or 4 amino acids of X 2 and X 3 are also contemplated by the invention and represented as X 13 wherein the lengths of 3 or 4 amino acids of X 2 and X 3 can be replaced by 5-aminopentanoic acid.
  • X 13 is not properly an amino acid, it is used as a place holder in some SEQ ID NO.'s to indicate 5-aminopentanoic acid bonded to the adjacent or underlying amino acid for improved stability.
  • X 14 is a variable selected from any of X 1 , X 2 , X 3 , X 4> X 5 , X 6 , X 7> X 8 , X 9 , or X 10 .
  • X 11 Gaba ⁇ for X 2 X 3 or X 3 X 2 or Amino function is on ⁇ carbon X 2 X 3 or X 3 X 2 or X 3 X 3 or and not ⁇ carbon
  • W is not added because W is less stable than F or P as described in US 5,109,384 ⁇ isomeric to leucine and isoleucine but not found in proteins and formed in the deamination of lysine. Also called caprine.
  • the novel peptide constructs of this invention can also be used to treat autoimmune disease such as rheumatoid arthritis (RA).
  • RA rheumatoid arthritis
  • the peptide antigen(s) used to make the construct would be antigens with defined epitopes recognized for Human Leukocyte Antigens (HLA) genotypes associated with for example, RA, combined with a TCBL as described above to either suppress or redirect the immune response.
  • HLA Human Leukocyte Antigens
  • TCBL Human Leukocyte Antigens
  • Other regions of potential usefulness include polymorphic regions of the minor histocompatability antigens.
  • TCBL' s can be selected from, but are not restricted to the following LFA-3 or
  • Peptide G from the MHC II molecule, or Hu IL-IO (SEQ ID NO.28) (Gesser et al, Identification of functional domains on human interleukin 10 1997, Proc. Nat. Acad. ScL 94:14620; Lie et al., supra and Tomita et al., supra) may be selected for redirection of immune responses.
  • T cells As activated T cells normally express MHC molecules, another way of immunomodulation is to take advantage of the programmed pathway established by antigen addition. T cells which receive a signal from the TCR and the MHC I to CD-8 + cells undergo apoptosis without other costimulatory signals (Sambhara et ah, supra 1991, Science, 252:1424). Therefore, the TCBL, peptide E, (the ⁇ 3 domain amino acids 223- 229 of the human MHC I conserved region can be used along with the autoimmune epitope to form a peptide construct according to another embodiment of this invention.
  • the peptide constructs of this invention may be used as or in vaccines as therapeutic agents for treatment of autoimmune disease, such as RA.
  • the vaccines will be administered often but not always with an adjuvant and on a regular regimen such as weekly, biweekly, monthly, quarterly, semi- annually or annually by one of the following routes, ID, IM or Sub-cu and perhaps also as a cutaneous transdermal or nasal delivery vehicle in amounts of from 1-100, usually 10-50, micrograms per kilogram of body weight.
  • the TCBL's (P 2 ) used to form the peptide constructs of this invention will be selected from those for normal induction and modulation of immune responses, including those selected to effect redirection from ThI or Th2, including, for example, those that are known to be related and involved in the normal events of activation, namely, IL-2, IL- 10, IL-12, IL-4, IL-l ⁇ 163-171 (VQGEESNDK (SEQ ID NO.13)) ⁇ e.g., Bajpai et al, Immunomodulating activity of analogs of noninflammatory fragment 163-171 of human interleukin-lbeta 1998 Immunopharmacology, 38:237; Beckers et al., Increasing the immunogenicity of protein antigens through the genetic insertion of VQGEESNDK sequence of human IL-I beta into their sequence 1993, J.
  • Examples of antigens associated with autoimmune disease include, for example, those mentioned above, including the background discussion and those shown in the following, non-limiting, representative examples, as well as in the literature references cited herein, the disclosures of which are incorporated herein, in their entirety, by reference thereto. Additional examples, may also be found in the following literature (de Lalla et al., Cutting Edge: Identification of Novel T Cell Epitopes in LoI /?5a by Computational Prediction 1999, J. Immunol., 163:1725 (LoI p5 a allergen from rye grass); Gautam et ah, supra 1998, /. Immunol., 161:60); as well as many others available to one of ordinary skill in the art.
  • the collagen induced arthritis (CIA) mouse model effectively mimics human disease to allow testing and development of rheumatoid arthritis (RA) therapies.
  • a Ligand Epitope Antigen Presentation System (L.E.A.P.S.) peptide hetero-conjugate vaccine has been developed and evaluated that contains as one component an epitope of human Collagen type ⁇ , and it has been determined in the CIA model that this vaccine is capable of limiting disease progression, as demonstrated by pathology, histopathology and arthritis index score.
  • L.E.A.P.S. technology converts a small peptide containing a disease specific epitope into an immunogen by attaching it a T cell binding peptide (TCBL) and presenting it to an immune cell.
  • TBL T cell binding peptide
  • a peptide from human ⁇ 2 microglobulin (J) is used as a TCBL.
  • the antigen is also called an immunogen and it is able to induce an immune response wherein antigen can be used inter-changeably with immunogen
  • small antigens sometimes referred and better described as epitopes are unable to induce an immune response without being made larger, or having other properties changed to make them into immunogens e.g., by adding a TCBL.
  • Efforts to develop vaccines for RA and evaluate them in animal models of human disease have shown promise, but to date these efforts have met with only limited success.
  • Obstacles that have stood in the way of development of a successful RA vaccine include: 1) identification of the epitope or epitopes to elicit a protective therapeutic response, 2) selection of an appropriate delivery technology and 3) demonstration of an appropriate therapeutic response in different models of the same disease.
  • a specific targeted antigen preferably a peptide that acts the same in humans and an appropriate animal model with disease symptoms and pathology similar, if not identical to those seen in humans, are both important issues to consider when developing a vaccine for an autoimmune condition.
  • the antigen issue is achieved, in part, based on findings that collagen Type II from at least 2 species (chicken and bovine) can induce arthritis in mice and rats, and in the case of human collagen Type ⁇ , or peptides thereof, can be recognized by cells or antibodies from RA patients, CIA induced rodents or both.
  • the choice of the appropriate immunogen to specifically modulate an autoimmune response is critical to the success of a therapeutic vaccine.
  • Several protein epitopes from collagen type II have been identified as relevant for consideration as the appropriate immunogen for an RA vaccine.
  • antibodies to citrullated peptides have been identified in RA and RA is known to be associated with a PAD gene defect, the enzyme responsible for citrullation (i.e.
  • L.E.A.P.S. technology is an appropriate technology to deliver a human collagen type II epitope as a therapeutic vaccine in a murine CIA model of RA.
  • the L.E.A.P.S. technology has been used successfully to elicit beneficial immune responses in actual disease-challenge models and in several different strains of mice differing in their MHC backgrounds.
  • ThI and DTH responses to an HIV epitope, HGP30, in BALB/c mice include: A) ThI and DTH responses to an HIV epitope, HGP30, in BALB/c mice, B) protective immune responses against herpes simplex virus (HSV-I) challenge of Balb/C, C3H, A/J and C57BL6 mice as a prophylactic vaccine and C) immune response modulation resulting in useful therapeutic effects when used as a vaccine for experimental autoimmune myocarditis (EAM) model in A/J mice and in the CIA model of RA in DBA/1J mice.
  • EAM experimental autoimmune myocarditis
  • L.E.A.P.S. technology has not been evaluated with human cells or as yet in clinical studies, there have been more animal protection efficacy studies with the L.E.A.P.S. than the PadreTM and Ii- KeyTM technologies.
  • the demonstrated efficacy of L.E.A.P.S. vaccines in mice with different MHC backgrounds suggest that CEL
  • a therapeutic vaccine has several advantages over the current immunosuppressive therapies used to treat RA. Unlike anti-cytokine therapies, the vaccine develops a targeted, specific modulation of the pathogenic immune response. The targeted nature of the immunization should minimize the side effects and risks of infection that can occur from current cytokine antagonist therapy.
  • Current therapies are passive in nature, involve larger amounts of materials must be given frequently often in a clinic or hospital setting and are prone to be immunogenic after prolonged use and generate immune responses to the therapeutic agent especially if not of human origin or humanized which can neutralize the therapeutic agent. In contrast, a limited number of immunizations with a vaccine should be required to halt and prevent or possibly reverse the progression of RA.
  • a therapeutic vaccine for RA will be different from the classical anti-infection vaccines since it will be administered during the course of the disease, after disease signs can be diagnosed. The challenge will be to provide therapy as late as possible after initiation of the disease process and appearance of clinical symptoms.
  • Therapies for EAM induced by My-I such as monoclonal antibodies (for TNF- ⁇ or IL- l ⁇ ), anti-complement receptor, cobra venom or recombinant proteins such as IFN- ⁇ are effective only if given in the first week, during the induction phase but are ineffective when given by day 10 or later.
  • mice CEL-2000 therapeutic vaccine for collagen induced arthritis (CIA) where the first step was to identify a good animal model for testing the vaccine, which is the collagen induced arthritis (CIA) model in young (6-7) week old male DBA/1J mice. These mice received 2 injections of bovine collagen the first in complete Freund's adjuvant (CFA) on day 0 and then 3 weeks later on day 21, in Incomplete CFA. After the second collagen injection, the mice were evaluated daily for any joint swelling or redness. Each of the paws was scored on a 4 point scale (Arthritis Index (AI)) with respect to the number of digits with symptoms and the thickness of the paw measured, at least 3-4 times a week. Each mouse was weighed weekly.
  • AI Arthritis Index
  • the first study is the CIA model CEL-2000 therapeutic vaccination conjugate specificity effect on AI scores (study 4.1 see Fig. 1 and legend).
  • Controls include groups with induced disease but no therapy and groups of healthy mice without induced disease.
  • a therapy control of Enbrel (3mg/kG daily) was included.
  • mice received two treatments of 33nmoles (for CEL-2000 equivalent to 4.8mg/kG) of CEL-2000, (the same dose as used for the L.E.A.P.S. HSV vaccines), or other conjugates of the same collagen peptide used in CEL-2000 using the same ICBLs as in the HSV or EAM systems for comparison using a GMP grade ICFA adjuvant.
  • CEL-2000 treatment limited the progression of disease, as indicated by AI scores, which were reduced to a greater extent than was seen with other conjugates, CEL-2001 or CEL-2002, in the same rank order as seen for similar L.E.A.P.S. conjugates in the EAM or HSV systems.
  • Enbrel treatment given on a daily basis for the first 14 days was slightly more effective than the vaccine as this dose and timing. This suggests a larger dose of CEL-2000 might be more effective.
  • Treatment started when group score was 3.5. This was done to have all groups starting with same score, as opposed to the range between 2.5 and 3.5 seen in the first study. The same dose of Enbrel as was used in the first study but was given every other day for the entire study period of 28 days.
  • AI scores Joint thickening, redness, immobilization of joints in a digit or total paw as measured for each of the 4 paws in the CIA mouse model of rheumatoid arthritis was reduced.
  • FIG. 4 is a summary plot of the analysis performed cores of the 4 areas from the following groups: Control no disease; CIA disease: CIA Disease Enbrel daily for 14 days continued for 2 more weeks before termination: CEL-2000 and CEL-2001 groups (vaccinated on day 0 and 7) from study 1 (Fig. 1) and from study 2 (Fig. 2) IX and 3X CEL-2000 vaccinated groups on day 0 and 7.
  • L.E.A.P.S. vaccines are effective therapeutic vaccines and based on other data the action does not appear to involve antibodies.
  • ICBL immune cell binding ligand
  • the outcome of the immune response to vaccines is likely at least in part determined by the cytokines elicited by the immunization.
  • Cytokines elicited by immunization of A/J mice with either the J, JgD or JH peptides emulsified in Seppic ISA51 were evaluated from serum obtained on days 3, 10 and 24 after immunization using Ray Biotech protein microarrays.
  • the cytokine profiles of serum obtained from mice immunized with either the JgD or the JH vaccines contained very similar results which were different from the adjuvant alone or the unmodified J ICBL vaccinated control mice.
  • By the third day after immunization there were increases in IL12p70, RANTES, GMCSF, MCP5, MCPl, IL9, and IL17 but not TNF- ⁇ levels.
  • IL12 is produced by DCs and macrophages and promotes the development of ThI responses which are characterized by production of IL2 and IFN- ⁇ .
  • a major goal of the L.E.A.P.S. vaccines has been to establish an ex vivo tissue culture system to study the effects and optimize the structure of the vaccines. It has been realized after many attempts with murine spleen cells, that these spleen cells are down regulated and or respond poorly to J-L.E.A.P.S. vaccines in tissue culture. Recent ex vivo studies with bone marrow cells indicate that two different J-L.E.A.P.S. vaccines (JH and JgD) can initiate and direct the nature of the subsequent immune response by production of IL12 but not TNF- ⁇ . It is the type of response that would counteract an antibody initiated hypersensitivity such as RA without exacerbating the condition by also stimulation production of TNF- ⁇ . Examples for CEL-2000:
  • CEL-2000 should be evaluated on purified human dendritic cells (DCs). Differences in the nature of the mouse and human immune responses, especially DCs, can influence the outcomes of vaccine administration.
  • the ability of CEL-2000 to elicit a response and cause the secretion of relevant cytokines in ex vivo studies of murine and human cells would provide the means to standardize treatment, evaluate some parameters of toxicity and help to define the mechanism of action of CEL-2000.
  • One antigen with defined epitopes recognized for rheumatoid arthritis is collagen type II including the peptide from position 254-273, (Krco et ah, "Characterization of the antigenic structure of human type II collagen", J Immunol, 1996 Apr 15;156(8):2761-8; Myers et ah, "Characterization of a tolerogenic T cell epitope of type ⁇ collagen and its relevance to collagen-induced arthritis", J Immunol., 1992 Aug 15; 149(4): 1439-43; Baldwin et al, "Structure of cDNA clones coding for human type II procollagen.
  • the alpha 1(11) chain is more similar to the alpha 1(1) chain than two other alpha chains of fibrillar collagens", Biochem J. 1989 Sep l;262(2):521-8).
  • collagen type II 254-273 is shown below.
  • the improved variants of collagen type II 254-273 are as follows. X 1 OXIXIX 4 X 7 XIX 3 XIXIXOX 4 XIX 2 X 9 XIXTX 4 XIX 2 X 7 (SEQ ID NO. 837) or
  • collagen type II 390-402. Other various antigens often with defined epitopes recognized for rheumatoid arthritis (RA) are collagen type II 390-402.
  • RA rheumatoid arthritis
  • Peptides from Type II human collagen (Krco et al. (1999 Identification of T cell determinants on human type II collagen recognized by HLA-DQ8 and HLA-DQ6 transgenic mice. J Immunol.163(3): 1661-5) referred therein as Peptide G 54-73 is shown by the SEQ ID NO. 487.
  • Peptide K at positions 94-113 is shown by SEQ ID NO. 490 (Krco et al, id.).
  • GLDGAKGEAGAPGVKGESGS SEQ ID NO. 490
  • Peptide 44 at positions 554-573 is shown by SEQ ID NO. 495 (Krco et al, id.).
  • the protein Osteopontin (OPN) contains a peptide referred shown by the SEQ ID NO: 1
  • Osteopontin (OPN) are shown as follows. X 10 X 3 XIX 6 XIX 3 X 4 (SEQ ID NO. 501) or
  • a peptide naJPl is indicated by SEQ ID NO. 503 (Prakken et al. (2004), Epitope- specific immunotherapy induces immune deviation of proinflammatory T cells in rheumatoid arthritis, Proc Nat Acad Sci USA 101:4228-4233).
  • a peptide dnaJPv is indicated by SEQ ID NO. 506 (Prakken et al, id.).
  • Type I diabetes is an autoimmune condition that most likely involves the THl pathway or if CTL is contemplated then the Tl pathway. Redirection of an antigen specific immune response from the THl to TH2 pathway may result in a non-diabetic condition.
  • One TCBL that is known to re-direct the THl to TH2 pathway is peptide "J".
  • the peptide "J" can be conjugated to a specific antigen wherein the conjugated construct can direct the immune response to the peptide antigen toward the THl pathway.
  • One example of such a construct is the L.E.A.P.S. construct.
  • the TCBL peptide "G” or the improved version "derG” can direct the immune response down the TH2 pathway.
  • the directed response is based upon data from several systems using peptide antigens from HSV, HIV, malaria, TB and murine myosin for antigen specific induction of antibody isotypes, DTH, and cytokine secretion (IL2, IL4 and IFN- ⁇ ).
  • the basis for the directed response is also founded on observed protection upon pathogen challenge as well as immune cell types evaluated to demonstrate effects of treatment with L.E.A.P.S. constructs.
  • one TCBL sequence contemplated in the L.E.A.P.S. construct of the present invention is the peptide "G" shown by SEQ ID NO. 15.
  • NGQEEKAGVVSTGLIGGG SEQ ID NO. 15
  • One variant of the peptide "G” is the compound known as "derG” with a triglycine spacer linked to the N terminus of the antigens having the sequence shown by SEQ ID NO. 50.
  • TCBL' s and spacers are also contemplated by the invention such as ICAM-
  • VLWKKQKDKVAELENSE (SEQ ID NO. 4)
  • TNF peptides known to activate macrophages are amino acids 70-80 as shown in SEQ ID NO. 5 (Britton et ah, A Tumor Necrosis Factor Mimetic Peptide
  • PSTHVL ⁇ TI SEQ ID NO. 5
  • the TCBLs contemplated by the invention can be selected from any of the sequence above but are not restricted to just the LFA-3 or FasL sequences described above (Lie et al. or Tomita et al., supra).
  • another TCBL can be the Hu IL-IO aa 152 - 16 o shown by SEQ ID NO.
  • TCBL sequence contemplated by the invention is the ⁇ 3 domain amino acids 223-229 of the human MHC I TCBL peptide E with a spacer. The sequence is shown by SEQ ID NO. 21.
  • the IL-l ⁇ at positions 163-171 shown by SEQ ID NO. 13 can also be used to form a peptide construct according to another embodiment of this invention (Bajpai et ah, supra; Beckers et ah, supra; Fresca et ah, supra; Antoni et ah, supra).
  • VQGEESNDK (SEQ ID NO. 13)
  • constructs containing spacers are also contemplated in additional embodiments.
  • a TCBL for the homologue of the C-terminal of human IL-IO (AYMTMKIRN), which is equivalent to SEQ ID NO. 18, is shown by SEQ ID NO. 763 (Kurte et al., "A
  • the improved versions of many of the sequence of the invention can contain multiple glycines 3 to 6 glycines in length.
  • carbodiimides are commonly used to modify proteins on their carboxylate groups. They react with proteins at about pH of 5 to 6 and can be used either alone or in combination with amines to create new amide bonds off the carboxyl. The reaction producing various new adducts are well known within the art.
  • a triglycine with or without one or two serines to separate two portions of the conjugate can be made by carbidiimide modification.
  • the triglycine with a single serine is shown by the SEQ ID NO. 7. GGGS (SEQ ID NO. 7)
  • MBS m-maleimidobenzoyl-N-hydroxy- succimide ester
  • EDC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide
  • N- isocyano-ethylmorphlin, bis-diazotized-benzidine, benzoquone and glutaraldehyde which are other reagents commonly employed to link polypeptides, can be employed in the present invention and are available from Pierce Chemical, Rockford, 111.; Eastman Kodak Chemicals, Rochester, N. Y.; Serva, Westbury, N.Y.; Sigma Chemical Co., St. Louis, Mo.; and E. Merck, Darnstadt, West Germany (Briand et al., Synthetic peptides as antigens: pitfalls of conjugation methods, 1985, J. Immunol.
  • the invention also contemplates formation of a single peptide from two smaller discontinuous peptides of IL-IO that are 11.2 Angstroms in length to form a TCBL to redirect from a THl to a TH2 in combination with, for example, the IDDM, PV or MS inducing epitopes (Reineke et al., supra).
  • Linkers of varying lengths to form a single chain may also be used.
  • GGGS SEQ ID NO. 7
  • GGGGS SEQ ID NO. 9
  • 1 or more repeats of the tetrapeptide or pentapeptide can be formed into the sequence shown by the following
  • RA Rheumatoid arthritis
  • TNF- ⁇ (TNF- ⁇ ) production is a problem and a major point of attack for new treatments.
  • the following peptide construct is prepared for use in the treatment of RA:
  • TNF-(X 70 -SO linker C-H 254 -I 73 (SEQ ID NO. 839) where the peptide TNF- ⁇ 7 o- 8 o is known to activate macrophages (10) and the collagen type ⁇ peptide C-II 254 - I73 (TGGKPGIAGFKGEQGPKGEP (SEQ ID NO. 836)) is an epitope associated with RA.
  • this peptide construct will be useful to achieve abortative T cell modulation since the improper sequence of events occurs with a binding that precludes the normal activation process.
  • TNF- ⁇ antagonist DFLPHYKNTSLGHRP (SEQ ID NO.24) (Chirinos-Rojas et al, supra) as follows.
  • TNF- ⁇ antagomst p ep tl de spacer C-H 254-273 (SEQ ID NO. 840)
  • peptide G or a derivative thereof from MHC-II ⁇ 2(135-149) to redirect the immune response from a THl to a TH2:
  • Example 2 prepared by utilizing the improved SEQ ID NO. 793-795, and its associated variants, as the TCBL, and the improved SEQ ID NO. 837 as the antigenic epitope.
  • Example 2 prepared by utilizing the improved SEQ ID NO. 793-795, and its variants as the TCBL, and the improved SEQ ID NO. 838 as the antigenic epitope.
  • the following peptide constructs are improved versions of SEQ ID NO. 852 prepared by utilizing the improved SEQ ID NO. 124 (peptide J), and its variants as the TCBL, and the improved SEQ ID NO. 837 as the antigenic epitope.
  • Example 7 The following peptide constructs are improved versions of SEQ ID NO. 852 prepared by utilizing the improved SEQ ID NO. 124, and its variants as the TCBL and the improved SEQ ID NO. 838 as the antigenic epitope.
  • Example 8 prepared by utilizing the improved SEQ ID NO. 793-795, and its associated varitants, as the TCBL, and the improved SEQ ID NO. 837 as the antigenic epitope, in a reverso form.
  • Example 8 prepared by utilizing the improved SEQ ID NO. 793, and its variants as the TCBL, and the improved SEQ ID NO. 838 as the antigenic epitope, in a reverso form.
  • the following peptide construct is an example of a variant of SEQ ID NO. 836 as the antigenic epitope and SEQ ID NO. 51 as the TCBL.
  • Example 11 prepared by utilizing the improved SEQ ID NO. 124, and its associated varitants, as the TCBL, and the improved SEQ ID NO. 837 as the antigenic epitope, in a reverso form.
  • Example 11 prepared by utilizing the improved SEQ ID NO. 124, and its variants as the TCBL, and the improved SEQ ID NO. 838 as the antigenic epitope, in a reverso form.
  • the following peptide construct is an example of a variant of SEQ ID NO. 50 as the TCBL and SEQ ID NO. 1 as the antigenic epitope as shown in SEQ ID NO. 197.
  • DGQEEKAGVVSTGLIGGGIAGFKGEQGPKGE SEQ ID NO. 197
  • Example 15 The following reverse peptide construct is an example of a variant of SEQ ID NO.
  • SEQ ID NO. 50 as the TCBL as shown in SEQ ID NO.
  • Example 16 The following peptide construct is an example of a variant of SEQ ID NO. 51 as the TCBL and SEQ ID NO. 1 as the antigenic epitope as shown in SEQ ID NO. 357.
  • Example 17 The following reverse peptide construct is an example of a variant of SEQ ID NO.
  • Example 18 The following peptide construct is an example of a variant of SEQ ID NO. 50 as the TCBL and SEQ ID NO. 487 as the antigenic epitope as shown in SEQ ID NO. 804.

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Abstract

La présente invention concerne des constructions de peptide, c’est-à-dire des polypeptides obtenus par liaison de deux peptides ou plus basés sur ou dérivés de molécules différentes, qui sont utiles dans le traitement ou la prévention de maladies auto-immunes, spécifiquement la polyarthrite rhumatoïde (RA) et des compositions contenant celles-ci, des procédés pour produire celles-ci et des procédés pour utiliser celles-ci ; les constructions de peptide étant de formule P1-x-P2 où P1 est un peptide associé à une maladie auto-immune, l’allergie, l’asthme, une réaction d’hôte contre greffe, la myocardite, le diabète, et une maladie à médiation immunitaire, qui se lie à un récepteur d’antigène sur un ensemble ou sous-ensemble de lymphocytes T ; P2 étant un peptide qui provoque une réponse immunitaire dirigée contre Th2 par l’ensemble ou le sous-ensemble de lymphocytes T auquel le peptide P1 est lié ou qui se lie à un récepteur de lymphocyte T qui amène l’ensemble ou le sous-ensemble de lymphocytes T auquel le peptide P1 est lié à initier, mais pas terminer, une réponse immunitaire amenant l’ensemble ou le sous-ensemble de lymphocytes T à subir une anergie et une apoptose ; et x étant une liaison directe ou un lieur pour lier de manière covalente P1 et P2.
PCT/US2009/037312 2008-03-14 2009-03-16 Procédés de préparation et composition de constructions de peptide utiles pour le traitement de la polyarthrite rhumatoïde WO2009114869A2 (fr)

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US9827181B2 (en) 2013-06-05 2017-11-28 Industrial Technology Research Institute Method and pharmaceutical composition for hair growth
US9827182B2 (en) 2013-06-05 2017-11-28 Industrial Technology Research Institute Method for promoting hair growth

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EP2254588A4 (fr) 2011-10-19
WO2009114869A3 (fr) 2010-09-23
US20110098444A1 (en) 2011-04-28
EP2254588A2 (fr) 2010-12-01
EP2254588B1 (fr) 2017-10-11
US11041013B2 (en) 2021-06-22

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